PROJECT SUMMARY. Pulmonary permeability edema (PPE) associated with pneumococcal pneumonia is a life-threatening condition, resulting from capillary barrier dysfunction in conjunction with impaired alveolar liquid clearance (ALC), with no proven treatment. Vectorial Na+ uptake mediates ALC. The identification of novel therapeutic approaches to simultaneously restore both ALC and barrier function represents a critical unmet need for pneumonia- associated PPE. Our main hypothesis is that the a subunit of the epithelial sodium channel (ENaC-?) represents a promising therapeutic target in PPE since it is a component of the highly-selective cation channel (HSC), which consists of ENaC-?, ? and ? subunits, and of the non-selective cation channel (NSC), which contains ASIC1a and ENaC-?. Both HSC and NSC channels mediate Na+ uptake. We also propose ENaC-? as a signaling molecule that strengthens barrier function in lung capillaries in the presence of pneumococci or their pore-forming toxin pneumolysin (PLY). Our main hypothesis is that ENaC-? exerts these actions mainly by blunting phosphorylation of the actin-binding protein filamin-A. In its non-phosphorylated form, filamin-A on the one hand promotes Na+ uptake capacity in sodium channels in alveolar epithelial cells and on the other hand it prevents stress fiber formation in capillary endothelial cells. Our novel hypothesis centers on the concept that ENaC-? functions as both an ion channel component and a signaling molecule, whose specific pharmacological activation restores ALC and barrier function during pneumococcal pneumonia. We will study the effect of specific activators of NSC (MitTx), of HSC (S3969 which binds to ENaC-?, not present in NSC) or of both (TIP peptide, which binds to ENaC-?), as well as genetic depletion or overexpression of ENaC-? on Na+ uptake capacity in pneumococci- or PLY-treated alveolar epithelial cells in vitro. We will moreover investigate whether ENaC-? activation or overexpression corrects S. pneumoniae- or PLY-induced barrier dysfunction in MVEC in vitro, through inhibition of Ca2+-dependent pathways that mediate filamin-A phosphorylation. Finally, we will test our hypothesis that direct ENaC-? activation is sufficient to mediate ALC and capillary barrier function during pneumococcal pneumonia in mice and in isolated perfused human lungs. Our expected outcomes include a better characterization of the unique role of ENaC-? in ALC during pneumococcal pneumonia, demonstration of a hitherto unknown role for ENaC-? in capillary barrier regulation and evaluation of the relative role of NSC versus HSC in protection from bacterial pneumonia-induced acute lung injury. Unraveling the unique mechanisms by which ENaC-? mediates ALC and barrier function during bacterial pneumonia can foster development of a novel breakthrough treatment for pulmonary permeability edema.